Porcine reproductive and respiratory syndrome isolates and methods of use

ABSTRACT

A method of predicting the virulence of a new or uncharacterized PRRS virus strain is provided wherein the strain is injected into swine and allowed to replicate for a period of from about 3-15 days. During this period, the rate of virus growth and/or the magnitude of viremia is determined, and this data is compared with a corresponding growth rate and/or viremia magnitude of a PRRS virus strain of known virulence, as a measure of the virulence of the new or uncharacterized strain.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is concerned with new isolated wild-type PRRSvirus strains and corresponding improved attenuated PRRS viruses, aswell as methods of measuring the magnitude of viremia, rate of growth,antibody response, and combinations thereof in such strains and viruses.More particularly, the present invention provides methods for predictingthe virulence of new or previously uncharacterized PRRS strains.

2. Description of the Prior Art

Porcine reproductive and respiratory syndrome virus (PRRSV) is anenveloped single stranded RNA virus classified in the familyArteriviridae (Cavanaugh, 1997). It causes a widespread disease of swinethat was first described as ‘mystery swine disease’ in the USA in 1987(Hill, 1990). The disease manifests as respiratory illness in all agegroups of swine leading to death in some younger pigs and severereproductive problems in breeding age females.

The dynamic nature of PRRSV allows for constant change in the diseaseand provides ample opportunity for the appearance of new strains(Andreyev et al., 1997; Murtaugh et al. 1998; Meng, 2000). The fact thatPRRSV changes so readily, coupled with its ability to cause devastatingproblems for swine producers, makes it an important subject for research(Mengeling et al., 1998; Pejsak et al., 1997) and for the development ofvaccines and other methods of reducing the effects of infection.Variation in levels of isolate virulence were demonstrated in lunglesions, and death in swine (Halbur et al., 1996), but efforts to linkbiological and immunological differences to specific genetic differenceshas been largely unsuccessful (Albina et al., 1998; Key et al., 2001;Yuan et al., 2001; Murtaugh et al., 2002; Grebennikova et al., 2004).Studies examining the safety and efficacy of PRRS vaccines include thework of Labarque et al., (2003), Mengeling et al., (2003a), andNodelijik et al. (2001). These studies show that under experimentalconditions, modified live PRRS vaccines reduce the amount and durationof viremia as well as fever and lung lesions after virulent challenge.

Opriessing et al. (2002) showed that isolates with high amino acidsequence homology in open reading frame 5 (ORF5) caused significantlydifferent levels of pneumonia in pigs. Variation in swine responses toPRRSV also are affected by host variation (Mengeling et al., 2003b).Virulence has been examined in relation to replication rates anddistribution of PRRSV in pigs (Haynes et al., 1997), to macrophagecopper clearing capabilities (Thanawongnuwech et al., 1998), and theanemia levels of the host animal (Halbur et al, 2002). However, thesemethods have been deficient in providing effective methods forpredicting the virulence of new or previously uncharacterized PRRSstrains.

Accordingly, what is needed in the art is a method of predicting thevirulence of PRRS strains. What is further needed in the art is a methodof predicting the virulence of PRRS strains based on the rate of in vivoPRRS viral growth and/or viremia magnitude in a swine afteradministration or exposure of the strain to a previously PRRS-freeswine.

SUMMARY OF THE INVENTION

The present invention overcomes the problems outlined above and providesmethods for predicting the degree of virulence of a new oruncharacterized PRRS virus strain. The methods generally involveassessing a new or previously uncharacterized strain of PRRS for atleast one of the following parameters: rate of viral growth; magnitudeof viremia; antibody response; or combinations thereof. The results ofsuch an assessment are then used to predict the degree of virulence ofthe new or previously uncharacterized strain. The methods generallyinvolve administering or exposing a PRRS-free swine to a quantity of thenew or uncharacterized PRRS strain and allowing the virus to replicatefor a period of up to about 15 days, more preferably from about 2-12days, still more preferably from about 3-10 days and still morepreferably from about 3-7 days. The mode of administration can beaccomplished using any conventional manner, including oral, intranasal,intramuscular, intra-lymph node, intradermal, intraperitoneal,subcutaneous, and combinations thereof, but most preferably throughintranasal administration. The amount of the dose for intranasaladministration is preferably up to about 5 ml, still more preferablybetween about 0.5 ml to about 4 ml, still more preferably between about1 ml and about 3 ml, and still more preferably about 2 ml. Theconcentration of virus in each dose should be up to about 5.0 Log₁₀TCID₅₀/ml, more preferably between about 1.0 to about 4.0 Log₁₀TCID₅₀/ml, and still more preferably about 2.5 to 3.5 Log₁₀ TCID₅₀/ml,and most preferably about 3.0 Log₁₀ TCID₅₀/ml. At a selected time ortimes during this replication period, biological samples are taken fromthe swine and measurements of the rate of growth of the administeredvirus, viremia magnitude, antibody response, and/or combinations thereofare taken. Data gathered from these measurements is then compared withthe rate of growth, viremia magnitude, antibody and/or combinationsthereof for a known and charaterized strain, as a measure of predictedvirulence of the new or unknown strain.

Using the methods of the present invention, the rate of PRRS viralgrowth, viremia magnitude, antibody response, and/or combinations ofthese characteristics were measured in swine which had one of eightdifferent PRRSV isolate strains administered thereto. Each of thesestrains had a known level of virulence and clinical diseasemanifestations. These same characteristics were also measured in swinewhich had a combination of all eight strains administered thereto.

More specifically, one hundred (100) healthy two-three week old pigswere divided randomly by weight into ten groups with each group havingten pigs. All pigs were tested for PRRS infection using HerdChek® PRRSELISA 2XR (IDEXX Laboratories Inc., Westbrook, Me.). Eight of the groupsreceived an administration of one of the eight strains, one groupreceived an administration consisting of a combination of all eightstrains, and the last group received an administration Eagle's MinimumEssential Medium (EMEM) to act as a control group. A sample of eachviral inoculation was retitrated for titer confirmation. Preferably thetiter of the administered virus is designed to mimic a natural exposurelevel of virus. Biological samples in the form of blood were collectedat various times throughout the 49-day experiment. Each sample wasanalyzed by virus isolation, quantitative reversetranscriptase-polymerase chain reaction (RT-PCR), HerdChek® PRRS ELISA2XR, and PRRSV protein-specific ELISA.

Virus isolation was performed on CL2621 cells by serially diluting serumand combining it with EMEM, gentamicin (Sigma Chemical Co., St. Louis,Mo.) and Fungizone (Invitrogen Corp., Grand Island, N.Y.). The dilutionswere then incubated and examined for cytopathic effect (CPE). TheReed-Muench calculation was used to determine titers.

RT-PCR was performed using the QIAamp Viral RNA Mini-Kit® (Qiagen, Inc.,Valencia, Calif.) and PRRSV was detected using a single-tube assay byTetracore, Inc. (Gaithersburg, Md.). To determine virus quantitation, astandard curve was developed and concentrations of the unknown sampleswere determined by linear extrapolation of the cycle threshold valuesplotted against the known concentration of the 3′ UTR transcriptproduct.

Antibody measurement using ELISA S/P ratios were generated usingHerdChek® PRRS ELISA 2XR using the manufacturer's instructions. PRRSVprotein-specific ELISA was performed using recombinant isolate VR2332nucleocapsid (N) and non-structural protein 4 (nsp 4) expressed in BL21(DE3)-RP cells (Stratagene, La Jolla, Calif.).

All pigs were weighed at the beginning and at the end of the study.Additionally, on every day of the study, each pig was evaluated andscored by a veterinarian for clinical signs of PRRS disease. Allresulting data was analyzed statistically and compared on agroup-by-group basis.

As used herein, “rate of growth” refers to the measurement of virusreplication over time in swine. Preferred examples of this measurementare provided in Example 1. “Viremia magnitudes” as used herein, refersto the concentration of virus circulating in the blood of swine.Preferred examples of this measurement are also provided in Example 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of mean serum virus titer versus time expressed asLog₁₀ TCID₅/ml for the swine test of Example 1;

FIG. 2 is a graph of mean PRRSV virus concentration in serum in theswine test of Example 1, as measured by real time RT-PCR;

FIG. 3 is a graph of mean S/P ratios versus time using a commercialELISA assay;

FIG. 4 is a graph illustrating repeated measures analysis for thecommercial ELISA assay and the Log₁₀ TCID₅₀/ml data of Example 1,wherein the group average under the ELISA S/P ratio curve was plottedagainst the group average area under the Log₁₀ TCID₅₀/ml;

FIG. 5 is a graph illustrating repeated measures analysis for thecommercial ELISA assay and RT-PCR concentration data of Example 1,wherein the group average area under the ELISA ratio curve was plottedagainst the group average area under the RT-PCR concentration curve;

FIG. 6 a is a graph of absorbance versus time for Example 1;

FIG. 6 b is a graph of absorbance versus time, illustrating the effectof PRRSV isolate or strain on nsp-4 IgG response, wherein the data arethe mean values of 10 animals, except where animals died;

FIG. 7 is a graph illustrating repeated measures analysis using thensp-4 and log₁₀ TCID₅₀/ml data of Example 1, wherein the group averagearea under the nsp-4 curve was plotted against the group average areaunder the Log₁₀ TCID₅₀/ml curve; and

FIG. 8 is a graph of repeated measures analysis using the clinicalscores and log₁₀ TCID₅₀/ml data of Example 1, wherein the group averageunder the clinical scores curve was plotted against the group averagearea under the Log₁₀ TCID₅₀/ml curve.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples set forth preferred isolates and procedures inaccordance with the present invention. It is to be understood, however,that these examples are provided by way of illustration only, andnothing therein should be deemed a limitation upon the overall scope ofthe invention.

EXAMPLE 1

Materials and Methods:

One hundred healthy 2-3 week-old pigs were obtained from a PRRS-freecommercial herd and were maintained at Veterinary Resources, Inc., Ames,Iowa, under the supervision of a veterinarian. Animals received food andwater ad libitum. All of the animal care and laboratory personnelinvolved with the study were blinded to the treatments given to thevarious groups of animals. Pigs were tested by HerdChek® PRRS ELISA 2XR(IDEXX Laboratories Inc. Westbrook, Me.) to determine if any pigs wereinfected with PRRSV. All of the pigs for this example tested negative.The pigs were then randomly divided by weight into 10 groups with 10pigs per group.

A total of eight PRRSV isolates were used in this example. Theseisolates have been designated VR-2332, Ingelvac® PRRS MLV, JA 142,Ingelvac® PRRS ATP, SDSU 73, Abst-1, MN 184, and 17198-6. These eightisolates span the history of PRRS disease, exhibit a wide range ofvirulence levels, and represent relevant clinical diseasemanifestations. All of the virus isolates grew readily on CL2621 cells(CL2621 is a proprietary cell line obtained from NVSL, Ames, Iowa) (anMA-104 monkey kidney cell line). Three of the primary field isolates,VR-2332, JA-142, and SDSU73, also had attenuated forms, Ingelvac®PRRSMLV (Boehringer Ingelheim Vetmedica Inc., St. Joseph, Mo.),Ingelvac® PRRS ATP (Boehringer Ingelheim Vetmedica Inc., St. Joseph,Mo.), and Abst-1, respectively. These attenuated forms all exhibit lowor undetectable virulence that was derived by in vitro passaging toattenuation. The PRRSV isolate ATCC VR-2332 was isolated in 1991 inMinnesota and was used at cell culture passage three. The attenuatedform of this virus is commercially available under the trade-nameIngelvac® PRRS MLV. The PRRSV isolate JA 142 (ATCC No. ______), providedby William Mengeling, National Animal Disease Center, Ames, Iowa, wasisolated in 1997 in Iowa from a severe “abortion-storm” case ofreproductive failure and was used at cell culture passage five. Theattenuated form of JA 142 is commercially sold under the trade-nameIngelvac® PRRS ATP and has been assigned ATCC No. VR-2638. PRRSV SDSU 73(ATCC No. ______) was recovered in Iowa from a severe case ofreproductive disease in 1996 and was used at cell culture passage one.The attenuated form of SDSU 73, designated Abst-1 (ATCC No. ______), wasobtained by 52 passages. The PRRSV isolate 17198-6 (ATCC No. ______) wasobtained from Oklahoma in 1997 from a herd experiencing severereproductive disease and was used at passage level four. The PRRSV MN184 isolate (ATCC No. ______) was obtained in 2001 from a swine farmexperiencing severe reproductive disease and sow mortality in southernMinnesota and was provided by Kurt Rossow, University of Minnesota, St.Paul. This isolate was used at a cell culture passage of one.Additionally, a pool consisting of a combination of all isolates wasproduced.

On day 0, each of the eight PRRSV isolates and the PRRSV pool werediluted to approximately 3.0 Log₁₀ TCID₅₀/ml in Eagle's MinimumEssential Medium (EMEM) (JRH Bioscience, Lenexa, Kans.) containing 4%FBS (JRH Bioscience, Lenexa, Kans.) and administered intranasally topigs at a dose of 2 ml (1 ml per nostril). The untreated control groupreceived 2 ml of media. The inocula were retitrated on 96-well platescontaining three-day-old CL2621 cells for titer confirmation using theReed-Muench method (Reed et al., 1938). The observed titers administeredto pigs, together with a description of the virulence level andisolation information, are shown in Table 1. TABLE 1 Virulence andInoculation Titer of Isolates. Titer log₁₀ Group Isolate Year IsolatedVirulence*** TCID₅₀/ml 1 VR 2332 1991 Moderate 3.43 2 Ingelvac ® USDAAttenuated 3.02 PRRS MLV* license 1996 VR2332 3 JA 142 1997 High 3.13 4Ingelvac ® USDA Attenuated 4.14 PRRS ATP* license 1999 JA 142 5 SDSU 731996 High 2.75 6 Abst-1* Attenuated Attenuated 4.18 1999 SDSU 73 7 MN184 2001 High 4.10 8 17198-6 1997 High 2.81 9 Pool** N/A High 3.71 10Control N/A N/A N/A*attenuated PRRSV isolates.**Mixture containing all of the eight isolates***Summary of lung lesions reported in Symposium on Emerging Diseases,Rome 2003.

The isolates were then compared to determine their genetic similaritythrough an analysis of their percent sequence identity. Sequenceidentity was determined by submitting virus samples to the University ofMinnesota Diagnostic Laboratory for sequence analysis. The results ofORF 5-6 were provided and then compared to a PRRS virus consensussequence. Individual base pair differences were noted and then the %sequence identity was compared between isolates. As those of skill inthe art are aware, blast searching can also be done at various websites.For example, the University of Minnesota provides a PRRSV database(ccgb.umn.edu/cgi-bin/common/web_blast.cgi) that lists sequences fromisolates from 1989-2003. Another frequently used site is the NCBI BLASTlink found at ncbi.nlm.nih.gov/BLAST.

As shown by the percent sequence identity and the dendogram in Table 2,the virulent field isolates are quite genetically distinct andrepresented a diverse group of PRRSV isolates. In contrast, the parentaland vaccine PRRSV pairs were nearly genetically identical. The pairwisecomparison and dendrogram of Table 2 were generated using the Lasergenesoftware suite of sequence analysis tools (DNASTAR, Inc, (Madison,Wis.)). TABLE 2 Pairwise comparisons of ORF5 nucleotide sequence ofvirulent and attenuated PRRSV isolates used in the study. Percentsimilarity is shown in the upper right and percent divergence is shownin the lower left of the table. The dendrogram shows the geneticrelatedness of the isolates. The bar indicates 1 nucleotide change per100 residues. VR2332 is the parent isolate of Ingelvac PRRS MLV, JA 142is the parent strain of Ingelvac PRRS ATP and SDSU 73 is the parentstrain of Abst-1. Percent Identity Ingelvac Ingelvac PRRS PRRS VR 2332MLV JA-142 ATP SDSU 73 Abst-1 MN 184 17198-6 VR 2332 99.7 91.0 90.5 90.089.6 86.4 90.4 Ingelvac 0.3 90.7 90.2 89.7 89.2 86.4 90.0 PRRS MLVJA-142 9.7 10.1 99.2 92.7 92.2 87.2 92.2 Ingelvac 10.3 10.7 0.8 91.991.4 86.4 91.4 PRRS ATP SDSU 73 10.9 11.3 7.8 8.8 99.5 87.2 91.7 Abst-111.5 11.9 8.4 9.4 0.5 86.7 91.2 MN 184 15.5 15.5 14.4 15.5 14.4 15.186.1 17198-6 10.5 10.9 8.8 9.7 9.0 9.6 15.9 Percent DivergenceEvaluation of Viremia

Blood samples were collected from each pig in each group by vacutaineron days 0, 1, 3, 7, 15, 21, 28, 35, 42, and 49. Serum was separated fromclotted whole blood by centrifugation at 3200×g for 20 minutes. Serumsamples were then divided for analysis by virus isolation, Log₁₀TCID₅₀/ml, quantitative reverse transcriptase-polymerase chain reaction(RT-PCR), HerdChek® PRRS ELISA 2XR, and PRRSV protein-specific ELISA.The serum samples in this study were processed immediately aftercollection and were chilled on ice within 3 hours of being obtained. Thesamples were stored for a maximum of 24 hr at 4° C. and at −70° C.thereafter. Serum tested by RT-PCR was frozen at −70° C. the day ofcollection and stored until the testing could be performed at which timeonly the number of samples that could be tested within 24 hours werethawed, extracted, and tested.

Virus isolation was performed on three-day-old CL2621 cells for samplescollected on days 0, 1, 42, and 49. One hundred μl of serum from eachpig on the remaining days of the study was diluted serially by ten-folddilutions to a final dilution of 10⁻⁶ in tubes containing 900 μl ofEMEM, 2% FBS, 50 μg/ml gentamicin (Sigma Chemical Co. St. Louis, Mo.),and 2.5 μg/ml Fungizone (Invitrogen Corporation, Grand Island N.Y.).Four replicates of each dilution were incubated on 96-well platescontaining CL2621 cells, at 37° C. and 4.5% CO₂ for eight days. Eachwell then was examined for cytopathic effect (CPE) and the titers weredetermined using the Reed-Muench calculation.

To obtain viral RNA for quantitative RT-PCR the QIAamp Viral RNAMini-Kit® (Qiagen Inc. Valencia, Calif.) was used as described in thekit instructions. A commercially available real-time, single-tube,RT-PCR assay for the detection of U.S. PRRSV was provided by TetracoreInc. (Gaithersburg, Md.) and used to detect PRRSV RNA. A minor groovebinding (MGB) 5′ nuclease probe and primers were designed by alignmentof GenBank isolates and based on conserved areas of the 3′ untranslatedregion (UTR). PRRSV RNA was reverse transcribed in a 25 μl single tubereaction consisting of Tetracore U.S. PRRSV Master Mix (18.9 μl Mastermix, 2 μl Enzyme mix 1, 0.1 μl Enzyme mix 2) and 4 μl of extracted RNA.The reaction tubes were loaded into the Smart Cycler II® block (Cepheid,Sunnyvale, Calif.) and software settings of fluorescent detection wereset for automatic calculation of the baseline with the backgroundsubtraction on. The thermal cycler program consisted of 52° C. for 1800seconds, 95° C. for 900 seconds, and 45 cycles at 94° C. for 30 seconds,61° C. for 60 seconds and 72° C. for 60 seconds. A PCR reaction wasconsidered positive if the cycle threshold (Ct) level was obtained at≦45 cycles. For quantitation, known amounts of serially diluted in vitrotranscript RNA product (1×10⁻¹ through 1×10⁸ copies/μl) were used togenerate a standard curve. Copy/ml concentrations of the unknown sampleswere determined by linear extrapolation of the Ct values plotted againstthe known concentration of the 3′UTR transcript product.

Antibody Measurement

ELISA S/P ratios were generated by performing the HerdChek® PRRS ELISA2XR according to the manufacturer's instructions. PRRSV protein-specificELISA for The HerdChek® was performed with recombinant isolate VR2332nucleocapsid (N) and nonstructural protein 4 (nsp 4) which wereexpressed in BL21 (DE3)-RP cells (Stratagene) from the plasmid pET 24bas fusion proteins containing an amino terminal myc-tag and a carboxylterminal 6× histidine tag. Denatured proteins were dialyzed in 0.1 MTris HCl, pH 8.0, 6 M guanidine-HCl, 2 mM EDTA and adjusted to aconcentration of 3 mg/ml. DTT was added to 300 mM and the solution wasfiltered through a 0.45 μm membrane. Reduced protein was added intorefolding buffer (100 mM Tris HCl, pH 8.0, 0.5 M L-arginine, 8 mMoxidized glutathione, 2 mM EDTA, 10 μM pepstatin A, 10 μM leupeptin, and1 mM PMSF), filtered (0.22 μm) and stirred overnight. The purifiedprotein was concentrated by tangential flow filtration (Pellicon XLUltracel PLC 5 kd, Millipore) and dialyzed against 20 mM Tris HCl, pH8.0. Proteins were analyzed on an Agilent 2100 Bioanalyzer with theProtein LabChip. Purified protein solutions were stored at −80° C.

Protein-specific ELISAs were performed by coating microtiter plates with100 ng recombinant protein in carbonate buffer, pH 9.6, or with bufferalone. Plates were blocked with 2.5% nonfat dry milk inphosphate-buffered saline containing 0.1% Tween 20 (PBST). One hundredμl of a 1:2000 dilution of serum was applied to duplicate wells for 2hours, after which plates were washed with PBST and antibody binding wasdetected by incubation with horseradish peroxidase-conjugated goat-antiswine IgG (heavy+light chains (KPL, Gaithersburg Md.) diluted 1:5000 for1 hour, followed by washing and color development with 100 μl of TMBsubstrate (KPL). Reactions were stopped with 1 M phosphoric acid andplates were read at 450 nm.

Body Weights

All pigs were weighed on day 0 (first day of study) and day 49 (end ofstudy). Pigs were weighed on a portable electronic weigh-bar scalesystem Weigh-Tronix™ model 615XL, (Weigh-Tronix Inc., Fairmont, Minn.).The scale was calibrated using certified test weights prior to and aftereach use.

Clinical Scores

On every day of the study each pig was scored by a veterinarian forrespiratory signs, behavior, and coughing on a scale of one to four foreach clinical sign. A normal animal was given a score of three, maximumclinical illness was a score nine and a dead animal received a score of12. Samples from all animals that died in the study were submitted tothe Iowa State University Veterinary Diagnostic Laboratory forpathological examination.

Statistical Analysis

All data were imported into SAS version 8.02 for data management andanalysis. Summary statistics including mean, standard deviation standarderror, median and frequency distributions were generated for all outcome variables as appropriate. Weight, RT-PCR, and Log₁₀ TCID₅₀/ml datawere analyzed by one way ANOVA for overall differences among thetreatment groups with pairwise testing for differences between treatmentgroups by Least Significant Difference t test. All tests for differencesbetween groups were designed as two-sided tests. Differences wereconsidered statistically significant at p≦0.05.

Some changes were made to the data to facilitate correlation analyses.The Log₁₀ TCID₅₀/ml values listed as <2.00 were set to 1.0. NegativeRT-PCR values were set to 1.0 and all RT-PCR values were normalized bytransformation to log base 10 before analysis. Control group resultswere not included in the correlation analyses. Results for each pig wereconverted to an approximate area under the curve using trapezoidal rule.Area under the curve was computed for the entire study period, from thefirst observation to day 15, and from day 15 to the last observation,although only the entire study period is shown in the figures.

Results

Virus Isolation and Log₁₀ TCID₅₀/ml Quantification

Before exposure on the day of infection no animals tested positive forPRRSV. At 1 day after intranasal infection, only 13 animals in 5 groupstested positive for virus. However, at 3 days after infection allanimals that were infected with field isolates, except for isolate17198-6, were virus positive with mean log₁₀ TCID₅₀/ml values rangingfrom 2.1 (SDSU-73) to 3.9 (MN 184). By contrast, animals inoculated withattenuated isolates were uniformly negative by cell culture. Theseresults are provided in FIG. 1. Peak levels of viremia, from 3.6 to 4.6log₁₀ TCID₅₀/ml were attained on day 7 for four of five virulentisolates and titers remained near or above 2 Log₁₀ TCID₅₀/ml in allvirulent virus groups for 21 days except for JA 142-infected pigs whichhad titers below that level.

The levels of viremia in the pigs inoculated with attenuated PRRSVisolates were lower than in pigs inoculated with virulent fieldisolates. The Abst-1 isolate, with the exception of day 3 postinoculation, was never re-isolated. Ingelvac® PRRS MLV viremiafluctuated between 0.5 and 1.0 Log₁₀ TCID₅₀/ml from days 7 to 28, andIngelvac® PRRS ATP varied between 0.4 and 1.2 Log₁₀ TCID₅₀/ml from days7 to 28. Attenuated isolate viruses were not recovered from serum afterday 28, and virus was recovered from only two of the virulent fieldisolate groups, the pool-infected and MN 184-infected pigs through day35 (also shown in FIG. 1). Nearly all pigs were nonviremic by virusisolation at days 42 and 49.

Overall, the more virulent isolates were observed to replicate fasterand to higher titers in pigs than were the attenuated isolates. Pigsinfected with the MN 184 isolate, in particular, showed a very rapidincrease in virus replication beginning before day 3 and reaching a peakof over 4.5 Log₁₀ TCID₅₀/ml on day 7. After peaking, the MN 184 viremiasteadily decreased but still maintained a significantly higher titer(t-test, p≦0.05) than all other isolates on days 28 and 35. A similartrend was observed in all of the remaining virulent groups, namelyVR2332, JA 142, SDSU 73, and the pool (see, FIG. 1). Pigs infected with17198-6 followed the same general trend described for the MN 184infected group but not as closely.

Groups of pigs administered the attenuated isolates (Ingelvac® PRRS MLV,Ingelvac® PRRS ATP, and Abst-1) followed a different trend. They showeda moderate increase in viral titer beginning after day 3 that reached apeak between days 7 and 15 at a viral titer more than a log less thanany of the virulent exposure groups and several orders of magnitude lessthan the MN 184-infected group. The titers observed in these attenuatedexposure groups then declined to zero on or before day 35 (See FIG. 1).

Virus Quantification by Real Time RT-PCR

Levels of viremia were also determined by real time RT-PCR since it waspossible that growth on CL2621 cells was not the same for all strainsand because RT-PCR might be a more sensitive measure than growth oncells for viremia. As shown in FIG. 2, virulent exposure groups showed adramatic increase in average concentration on day 1 and all groupspeaked above 8 logs/ml between days 7 and 15. The virulent exposuregroup concentrations then gradually tapered off through the next severalweeks, reaching concentrations below 4 logs/ml by day 49.

The attenuated strain exposure groups showed a much less dramaticincrease in concentration that also began around day 1 and the averagegroup titer never reached or exceeded 7 logs/ml (FIG. 2). Theconcentrations observed for the attenuated exposure groups weremaintained at fluctuating levels showing a wide range in values in theweeks following the exposure. The fluctuations were primarily due tosporadically high values in a single pig. The three attenuated strainexposure groups all peaked on different days of the study. The Ingelvac®PRRS MLV group peaked at a concentration of 4.31 logs/ml on day 28, theIngelvac® PRRS ATP group peaked at 6.58 logs/ml on day 3, and the Abst-1group peaked at 6.85 logs/ml on day 35, which was the highest titerachieved by an attenuated isolate (FIG. 2). Additionally, the averageconcentration of the virulent isolate groups were observed to besignificantly higher (P<0.05) than the average concentration of theattenuated strain groups on days 3 and 15, but on day 49 the averageconcentration of the virulent isolate groups was significantly lower(P<0.05) than that of the attenuated isolate groups.

HerdChek® PRRS ELISA 2XR

As shown by FIG. 3, the humoral immune response to PRRSV, as measured byHerdChek® PRRS ELISA 2XR S/P ratios, showed that the virulent isolateexposure group averages rose above the 0.4 cutoff for a positive resulton day 15. By contrast, the attenuated strain exposure group averageswere negative and all three groups remained below 0.4 until after day21. The Ingelvac® PRRS MLV and Ingelvac® PRRS ATP groups showed positiveresults on day 28, but the Abst-1 group did not show an average S/Pratio over 0.4 until day 42.

In comparing the humoral response of groups infected with virulentisolates or the pool to groups inoculated with attenuated strains, itwas clear that the kinetics and magnitude of the antibody response wasassociated with the level of viremia, particularly between 14 and 35days after infection. This observation is further supported by thecorrelation between viremia levels and humoral antibody responsesdetermined by paired comparisons of HerdChek® PRRS ELISA 2XR S/P ratiosto either virus titration or RT-PCR. FIGS. 4 and 5 show that the humoralantibody response is closely associated with viral load over the entirestudy period with a correlation coefficient r=0.858 for virus titrationand r=0.794 for RT-PCR. These associations were highly significant(p<0.0001 in each case). Moreover, attenuated strains show low antibodyresponses and viral loads, whereas virulent strains show high responses.

PRRSV Protein-Specific ELISA

To gain additional insight into the relationship between differences inPRRSV inocula and humoral immune responses, the antibody titers againstN, the major structural protein, and nsp 4, an essential but minornonstructural protease, were determined. FIG. 6 a illustrates that thekinetics of the nucleocapsid anti-N IgG response were nearly identicalin all groups of pigs, with a peak titer on day 28 followed by a sharpdecline in the next 7-14 days, after which the levels were maintained orrose slightly between days 42 and 49.

The magnitude of the response for each strain was similar to that foundin the HerdChek® PRRS ELISA 2XR results, and consistent with the levelsof viremia. The lowest peak titers at day 28 were observed in the groupsinoculated with attenuated strains, and the highest titer was attainedin pigs infected with the highly virulent MN 184 isolate. By day 49 theanti-N titer was equivalent in all groups except for MN 184 and thepool, suggesting that the humoral response to MN 184 may bequalitatively different. Additionally, only 5 pigs survived to day 49 ineach of these two groups, which is reflected in the increased standarderror at day 49 in the MN 184 group.

As shown by FIG. 6 b, the IgG response to nsp 4 was substantiallydifferent than to N. No anti-nsp 4 antibody was detected before day 21,the overall response was much weaker, and no significant response wasdetected in the groups receiving Ingelvac® PRRS MLV and Abst-1.Moreover, the magnitude of the anti-nsp 4 response was not associatedwith level of viremia. The responses to VR 2332, JA 142, MN 184, and thepool were all equivalent, with a peak at day 28, followed by a declineat day 35, then rising again at day 42, whereas the magnitude, timecourse and duration of viremia varied among these four groups. FIG. 7illustrates that when examining the repeated measures analysis, data forthe nsp 4 ELISA compared to the Log₁₀ TCID₅₀/ml data, it can be seenthat there is no correlation between level of viremia and nsp 4 humoralantibody response.

Body Weight

There was no significant difference in the mean weight of any of thegroups on day 0 of the experiment (P=0.099). On day 49 pigs inoculatedwith the attenuated strain Abst-1 had the highest mean weight, which wassignificantly higher then all other groups except for the control group(Table 3). Also, on day 49, the mean weights of all the virulent isolateexposure groups except for the 17198-6 group were significantly lowerthan the control group (Table 3). The mean weights of the attenuatedstrain exposure groups Ingelvac® PRRS MLV and Ingelvac® PRRS ATP and thecontrol group were statistically equivalent (Table 3). TABLE 3 AverageBody Weights. Isolate Day 0 Day 49 VR 2332 6.38¹ 33.5^(b) Ingelvac ®PRRS MLV 6.56 34.6* JA 142 6.42 32.7^(b) Ingelvac ® PRRS ATP 6.24 35.0*SDSU-73 6.59 32.9^(b) Abst-1 6.69 39.4^(a) MN 184 6.73 23.7^(c) 17198-66.36 34.5* Pool** 6.51 23.0^(c) Control 6.48 38.4*¹Weights are in kg. There were no significant differences in mean wt atday 0.*Indicates statistically equivalent weights among these groups on day49.**Pool was a mixture containing all eight isolates.^(a)Significantly greater than all groups except the Control group (p ≦0.05).^(b)Significantly less than the Control group.^(c)Significantly less than all other groups.

Clinical Scores

Increases in average clinical scores were observed in only four of thevirulent exposure groups: JA 142, SDSU 73, MN 184, and Pool. Thesehigher scores were maintained throughout the study while the remaininggroups, both virulent and attenuated exposures, had essentially normalclinical scores for the duration of the study. The only major cause ofchanges in the average clinical scores observed in this study occurredwhen one or more animals died in the associated treatment group (Table4). TABLE 4 Mortality of Pigs after Exposure Group Strain MortalityDay(s) of Death(s) 1 VR 2332 0/10 N/A 2 Ingelvac ® PRRS MLV 0/10 N/A 3JA 142 1/10 = 10% 17 4 Ingelvac ® PRRS ATP 0/10 N/A 5 SDSU 73 2/10 = 20%9, 23 6 Abst-1 0/10 N/A 7 MN 184 5/10 = 50% 14, 14, 17, 23, 41 8 17198-60/10 N/A 9 Pool** 5/10 = 50% 12, 16, 17, 21, 21 10 Controls 2/10* 41, 48Attenuated PRRSV 0/30 = 0% Virulent PRRSV 13/60 = 22%All deaths in treatment groups were attributed to moderate or severenon-suppurative interstitial pneumonia due to PRRSV with secondarybacterial infection.*Deaths attributed to bacterial pneumonia with no PRRS involvement.**Pool was a mixture containing all eight isolates.

The severity of clinical disease was highly associated with viral load(p<0.001 for virus titration). As shown in FIG. 8, the clinical scoreswere highest for the groups infected with MN 184 and the Pool. Fiftypercent of the pigs in each group died, and virus titration indicatedthat the level of infection was substantially higher than for all othergroups. The differences in viral load as determined by RT-PCR were lessmarked (data not shown) and the correlation of clinical signs with viralload by RT-PCR was less than with virus titration (r=0.556 versusr=0.803, respectively). The clinical scores in group 10 (Control)increased after the death of two pigs from bacterial pneumonia. Bothpigs were shown to be PRRSV negative by immunohistochemical staining oflung tissue, negative virus isolation and real-time PCR analyses, andthe complete lack of seroconversion by HerdChek® PRRS ELISA 2XR orprotein-specific ELISA. Later findings indicated that various bacterialpathogens were present in animals that died unexpectedly during thestudy these deaths were likely attributed to secondary bacterialinfection (Table 5). TABLE 5 Cause of Mortality after Exposure Pig #Group Cause of Death Day of Death 993 JA 142 PRRS & Streptococcus suis17 948 Neg Control Arcanobacterium 41 pyogenes & Pasteurella multocida983 Neg Control A. pyogenes & P. multocida 48 922 SDSU 73 PRRS andbacterial pneumonia* 9 918 SDSU 73 PRRS and Escherichia coli 23 973 MN184 PRRS and Actinobacillus suis 14 992 MN 184 PRRS and A. suis 14 980MN 184 PRRS and E. coli 17 971 MN 184 PRRS and E. coli 23 958 MN 184PRRS and E. coli 41 976 Pool PRRS and A. suis 12 970 Pool PRRS and V S.suis 16 972 Pool PRRS and S. suis 17 995 Pool PRRS and S. suis 21 969Pool PRRS and A. pyogenes 21*The diagnostic report indicated “bacterial pneumonia” with no specificagent listed.Discussion

One objective of this example was to examine various PRRSV isolates withknown levels of virulence to determine if there was a relationship within vivo replication that could be used to predict the virulence of PRRSVisolates without the necessity of performing controlled challengeexperiments. Additionally, it was of interest to determine therelationship between isolate virulence, levels of viremia, and thehumoral antibody response. Finally, it may be of interest to developvaccines against the PRRSV isolates that are found to be virulent usingthe methods of the present invention. It would be a goal to have suchvaccines provide some degree of protection against other virulentisolates; however, such cross-effectiveness may not be universal for allPRRSV isolates and further testing would be required. However, it isclear that the present invention provides an effective tool foridentifying prime candidates for vaccine development.

In order to test PRRSV isolates under the same conditions it wasnecessary to use dosages of licensed vaccines that were below theminimum immunizing dose established with the USDA and that were notrepresentative of a commercial dose. Also, the intranasal route ofadministration of the MLV vaccines used in the study was not inaccordance with the USDA label and was only used to mimic a more naturalexposure. The typical commercial dose of the modified live PRRS vaccines(Ingelvac PRRS and Ingelvac PRRS ATP) is much higher than what was usedin this experimental trial. These experimentally low doses of modifiedlive PRRS vaccine do not represent the actual product dosage and formused in the field and readily explains the reported serologicalresponse. Using a commercial dose of vaccine, serological titers asmeasured via the IDEXX assay would be detectable by day 14. In thistrial using titers of approximately 3 logs, this serological responsewas delayed and lower. This was to be expected, but was done to insureconsistency of titer administration between groups and to facilitate theanalysis and comparison between virulent and attenuated isolates.Although not specifically addressed in this Example, the effect of doseis likely much more significant for an attenuated or less virulent virusthan it is for a virulent field virus that can quickly grow in and berecovered at over 4 logs/ml in pig serum within 3-7 days of exposure.The higher recommended intramuscular commercial dose gives HerdChek®PRRS ELISA 2XR S/P ratios above the 0.4 cutoff by 14 days postvaccination which is one-half the amount of time observed for the dosesused in this study (Roof et al., 2003). The nominal dose used in thisstudy, 2×10³ TCID₅₀ per animal, caused 50% mortality in groups thatreceived isolate MN 184, and anti-nucleocapsid responses in all groups.Higher doses were not tested since excessive mortality in groupschallenged with highly virulent strains would have compromised the studyobjectives. In addition, previous studies had shown no difference inclinical signs and viremia in young pigs inoculated with PRRSV isolateVR2332 at doses of 10^(2.2), 10^(3.2) and 10^(4.2) TCID₅₀ per animal.

Both the Log₁₀ TCID₅₀/ml and real time RT-PCR results showed that theviremia levels vary significantly among groups following PRRSV exposure.This indicates that the growth rate of PRRSV in pigs is a phenotypiccharacteristic of the virus independent of possible variation in pigsusceptibility to infection. In addition, attenuation of PRRSV byadaptation to growth on CL2621 cells reduced not only its ability togrow in pigs, but altered the kinetics of viral replication so that peakviremia occurred at later times. A similar observation was also made byChang et al. (2002), who showed that even a limited period of cellculture passage of the moderately virulent PRRSV isolate VR 2332 reducedviral growth in pigs and delayed significantly the time to peak viremia.However, a delayed time to peak viremia is not diagnostic for in vitrocell culture passage or for attenuation, since the highly virulentisolate 17198-6 also showed a delayed time to peak viremia.

Overall, virulent isolates showed substantially higher viremia levels inserum than did attenuated strains at equivalent doses of inoculation.For example, the highest observed virus titer in any of the attenuatedisolate exposure groups was 1.22 logs on day 15 in pigs given Ingelvac®PRRS ATP, whereas the lowest titer of any virulent group on day 15 was2.40 logs in the SDSU 73 group. The peak of viremia at days 3-7 and thelevels of virus detected (all >3.5 logs/ml) was highly consistent amongvirulent PRRSV isolates, though MN 184 was significantly greater in itsmagnitude and duration, with virus titers still present on days 28 and35. This supports the concept that highly virulent PRRSV isolatesreplicate to a substantially higher titer in vivo than do attenuated orlowly virulent isolates, but they do not establish a direct quantitativerelationship between level of virulence and level or rate of in vivogrowth among wild-type PRRSV (Haynes et al. 1997).

The real time RT-PCR results were statistically very similar to theLog₁₀ TCID₅₀/ml results, indicating that both methods measure relativelevels of infectious virus among groups. The Pearson correlationcoefficient between the RT-PCR and Log₁₀ TCID₅₀/ml day 7 data was 0.89and for the average real time RT-PCR and Log₁₀ TCID₅₀/ml results was0.88. The concentration values determined by real time RT-PCR may havebeen several orders of magnitude higher than Log₁₀ TCID₅₀/ml values forseveral reasons, including differences between the frequency of viralparticles containing the target amplicon and particles that are fullyinfectious on CL2621 cells, and the presence of neutralizing antibodiesthat could lower infectivity (Dianzani et al., 2002). However,neutralizing antibody is unlikely to account for the difference, becauseit was observed at all time points, including times before which ananti-PRRSV antibody response had been produced.

The copies/ml values determined by real-time PCR were higher than theinfectious titer values measured in cell culture by TCID₅₀/ml. This isbecause a standard curve based on the copies of genome of the virus isroutinely used for quantitative PCR which directly amplifies a genomicsequence of the virus rather than known infectious virions. Biologicassays such as cell culture do measure the presence of infectivity,however, they may not count all of the infectious particles present in apreparation. Factors that could affect the infectious titer such as cellculture conditions and in vivo antibodies, which may neutralize virus,have been observed in other studies, underestimating the amount ofinfectious virus measured in TCID₅₀/ml in sera. Alternatively, somenon-infectious or replication-defective virus may be present which wouldbe reflected by higher copy numbers.

In general, the ELISA observations support the concept that themagnitude of the humoral immune response is related to the level ofviral replication during acute infection. The trend indicated in FIGS. 4and 5 illustrates this relationship. A slower and less intense humoralimmune response was triggered by the cell-culture attenuated virusisolates, whereas a faster and more intense humoral immune response wastriggered by the virulent isolates. In addition these observations alsodemonstrate that at least two factors, isolate type and infectious dose,impact relative S/P ratio values in the HerdChek® PRRS ELISA 2XR.Although the ELISA results shown in FIG. 3 indicate a clear positive ornegative average group response, it is important to note the variabilityamong individual animals. Some pigs within attenuated virus groups werepositive before day 21, and some pigs in the virulent groups remainednegative up to day 21.

Analysis of specific antibody responses to N and nsp 4 show that immuneresponses to PRRSV vary in intensity independently of the inoculatingisolate. Antibody responses to the N protein in animals that wereinoculated with the highly virulent isolates MN 184 and JA 142 showed atrend similar to that of all the isolates but to a higher magnitude.Pigs inoculated with MN 184 and JA142 also had the highest viral titers,as shown in FIGS. 1 and 2. This indicates that the level of humoralimmune response may be related to the viral load in acute infection asmeasured by viral titer. Interestingly, the time course of response wasthe same in all groups, even though the time to peak titer was delayedfor highly virulent strain 17198-6 and the attenuated strains. The nsp 4antibody response, by contrast, was low at all of the time points andfor all of the isolates, both attenuated and virulent. The time courseof anti-nsp 4 response was equivalent in all of the groups despitedifferences in the time to peak viral load among groups, as observed forthe anti-N antibody response. All pigs had low anti-nsp 4 responses asshown in FIG. 7.

These observations indicate that some of the PRRSV proteins elicit amore robust response from the host immune system regardless of exposureisolate virulence. However, the observations also indicate that themagnitude of the immune response to the more immunogenic proteins islikely related to the virulence of the exposure isolate, or the abilityof the isolate to replicate in vivo. It also is possible thatdifferences in antibody response might be due simply to geneticdifferences among isolates that result in differences in antigenicreactivity such that antibodies directed against N and nsp 4 of otherisolates do not react or react poorly to the recombinant proteinsexpressed from isolate VR2332 that were used to coat the ELISA plates.However, several lines of evidence suggest that the observed differencesin antibody levels reflect immunologically relevant responses. IsolateMN 184 shows the greatest genetic difference from VR2332, as determinedby ORF 5 comparisons, yet has the highest anti-N antibody response.Kapur et al. (1996) showed previously that relative differences amongPRRSV isolates in one open reading frame are also present in other openreading frames. Additionally, individual proteins contain conserved andnonconserved regions (e.g. Kapur et al., 1996) and extensive immunogenicreactivity may be directed toward the conserved epitopes (Ostrowski etal., 2002). Nevertheless, ELISA results based on antibody reactions withpurified PRRSV proteins may be affected by genetic and antigenicvariation, and these effects must be considered. Refolding ofrecombinant proteins was performed, but no differences were observedbetween ELISA plates coated with nonrefolded or refolded proteins.

It was noted that at approximately 4 to 5 weeks after inoculation, arelatively large decrease in the antibody response to both the N and nsp4 proteins occurred. A similar peak of 1 to 2 weeks followed by adecline of antibody reactivity was previously noted by Foss et al.(2002) for GP5, the major envelope glycoprotein. Taken together, theseobservations suggest that the response to individual viral proteinslikely does not represent the full picture of the pig's immune responseto PRRSV since the humoral immune response as measured by the HerdChek®PRRS ELISA 2XR does not show a similar transient peak of antibodyreactivity.

Reduced growth and mortality were the key correlates of virulence andviral in vivo growth rate. The lower mean weight observed in thevirulent isolate exposure groups most likely reflected a difference inthe ability of a PRRSV isolate to replicate in vivo and induce a moresevere illness in the pig. These observations are consistent withpreviously reported data that PRRSV infection may cause anorexia with a25 to 40 percent reduction in daily weight gain (Thacker, 2003). Theclinical scores of animals exposed to the virulent isolates showed rapidincreases shortly after the inoculation, whereas there was virtually nochange in the scores of the attenuated virus exposure animals. Thisincrease in clinical signs was reflected in the observed death rates of50%, 20%, and 10% in the virulent exposure groups receiving PRRSisolates MN 184, SDSU 73, and JA 142, respectively. In contrast, theattenuated exposure groups incurred no deaths. The relationship betweenrapid viral growth and viral pathogenesis under the same conditions ofviral exposure were most evident in comparing the groups exposed to MN184 and Abst-1. The inoculation titers were virtually the same, 4.10logs/ml and 4.18 logs/ml, respectively, and yet, as indicated in FIG. 8,there were remarkable differences in the way the two isolates affectedpigs. The Abst-1 isolate was nearly inert, it hardly replicated in vivoand caused no clinical signs. By contrast, the MN 184 isolate replicatedto extremely high titers in vivo and caused severe clinical signs,resulting in the death of 50% of exposed animals. Also notable, thegroup of pigs exposed to the pool of all virus isolates showed about thesame virological, clinical, and immunological responses as pigs exposedto MN 184. This finding indicates that the most rapidly replicatingvirus in a mixed infection is likely to outcompete other isolates sothat the net result is essentially the same as an infection with thesingle isolate having the highest growth potential.

The notable in vivo differences between virulent and attenuated PRRSVisolates shed light on the relationship between the virulence of anisolate and its in vivo growth and replication. When administered atequivalent doses in pigs, the more virulent isolates show Log₁₀TCID₅₀/ml titers and RT-PCR concentrations that are exponentially higherthan the attenuated isolates. The virulent isolates induce a more rapidand intense humoral immune response. The virulent isolates negativelyaffect weight gain and induce higher death rates and more severeclinical signs as compared to the attenuated isolates.

In conclusion, the example and tests in the present application indicatethat attenuated and virulent PRRSV isolates induce remarkably differentclinical signs, as well as immune responses that differ in intensity.These differences are attributed to the ability of the virus toreplicate in vivo, a phenotypic characteristic that can be measuredquantitatively in serum samples and may be developed for predicting thevirulence of PRRSV isolates.

REFERENCES

The teachings and content of each of the following references areincorporated by reference herein.

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1. A method of predicting the virulence of a PRRS virus strain ofunknown virulence, comprising the steps of administering a quantity ofsaid PRRS virus strain into PRRS-free swine, allowing the virus toreplicate in said swine for a period of from about 3-15 days, measuringthe rate of virus growth and/or the magnitude of viremia during saidperiod, and comparing said rate of growth or viremia magnitude with therate of growth and/or viremia magnitude of a PRRS virus strain of knownvirulence as a predictor of virulence of PRRS strain of unknownvirulence.
 2. The method of claim 1, said period being between fromabout 3 to 7 days.
 3. The method of claim 1, including the step ofmeasuring the magnitude of viremia during said period, and comparingsuch magnitude with the viremia magnitude of said known PRRS virusstrain.
 4. The method of claim 1, said quantity of administered virusbeing similar to the amount an animal would receive by natural exposure.5. The method of claim 1, said PRRS virus being adminstered by a methodselected from the group consisting of oral, intranasal, intramuscular,intra-lymph node, intradermal, intraperitoneal, subcutaneous, andcombinations thereof.
 6. The method of claim 1, said rate of growth orviremia magnitude being measured in a biological sample from said swine.7. The method of claim 1, said viremia being measured by Log₁₀TCID₅₀/ml, reverse transcriptase-polymerase chain reaction, PRRSspecific ELISA, PRRS protein-specific ELISA, and combinations thereof.8. The method of claim 1, further including the step of observing saidswine for clinical signs of PRRS infection after administration of saidPRRS strain.
 9. The method of claim 8, said clinical signs includingrespiratory signs, behavior, coughing, and combinations thereof.
 10. Themethod of claim 1, further including the step of predicting that thePRRS strain will be of high virulence when its rate of growth or viremiamagnitude are similar to that of a strain with high virulence.
 11. Themethod of claim 1, further including the step of predicting that thePRRS strain will be of low virulence when its rate of growth or viremiamagnitude are similar to that of a strain of low virulence.
 12. Themethod of claim 1, said administered amount of PRRS virus being up toabout 5 ml of inocula having a viral concentration of up to 5.0 Log₁₀TCID₅₀/ml.